Representative SRM chromatograms.

<p>Panel A: SRM chromatograms of a human blank plasma sample; Panel B: SRM chromatograms of a human blank plasma sample with IS added; Panel C: S/N of SN-38, SN-38G, CPT-11 and APC at the LLOQ (10 ng/mL for CPT-11 and 1 ng/mL for SN-38, SN-38G and APC); Panel D: SRM chromatograms of an extracted plasma sample of a treated patient showing IS, SN-38, SN-38G, CPT-11 and APC. The concentrations measured were 7.42, 16.57, 80.27 and 12.91 ng/mL for SN-38, SN-38G, CPT-11 and APC, respectively.</p

Development and Validation of a High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for the Simultaneous Determination of Irinotecan and Its Main Metabolites in Human Plasma and Its Application in a Clinical Pharmacokinetic Study

<div><p>Irinotecan is currently used in several cancer regimens mainly in colorectal cancer (CRC). This drug has a narrow therapeutic range and treatment can lead to side effects, mainly neutropenia and diarrhea, frequently requiring discontinuing or lowering the drug dose. A wide inter-individual variability in irinotecan pharmacokinetic parameters and pharmacodynamics has been reported and associated to patients’ genetic background. In particular, a polymorphism in the <i>UGT1A1</i> gene (<i>UGT1A1*28</i>) has been linked to an impaired detoxification of SN-38 (irinotecan active metabolite) to SN-38 glucuronide (SN-38G) leading to increased toxicities. Therefore, therapeutic drug monitoring of irinotecan, SN-38 and SN-38G is recommended to personalize therapy. In order to quantify simultaneously irinotecan and its main metabolites in patients’ plasma, we developed and validated a new, sensitive and specific HPLC–MS/MS method applicable to all irinotecan dosages used in clinic. This method required a small plasma volume, addition of camptothecin as internal standard and simple protein precipitation. Chromatographic separation was done on a Gemini C18 column (3 μM, 100 mm x 2.0 mm) using 0.1% acetic acid/bidistilled water and 0.1% acetic acid/acetonitrile as mobile phases. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring. The standard curves were linear (R² ≥0.9962) over the concentration ranges (10–10000 ng/mL for irinotecan, 1–500 ng/mL for SN-38 and SN-38G and 1–5000 ng/mL for APC) and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy, determined on three quality control levels for all the analytes, were always <12.3% and between 89.4% and 113.0%, respectively. Moreover, we evaluated this bioanalytical method by re-analysis of incurred samples as an additional measure of assay reproducibility. This method was successfully applied to a pharmacokinetic study in metastatic CRC patients enrolled in a genotype-guided phase Ib study of irinotecan administered in combination with 5-fluorouracil/leucovorin and bevacizumab.</p></div

Plasma concentration-versus-time profiles of CPT-11 and its main metabolites: SN-38, SN-38G and APC.

<p>Panel A: Plasma concentration-<i>versus</i>-time profiles of CPT-11, SN-38, SN-38G and APC in one patient receiving 310 mg/m² of irinotecan during the I and the II administration of the first therapy cycle; Panel B: Plasma concentration-<i>versus</i>-time profiles of CPT-11, SN-38, SN-38G and APC in one patient receiving 370 mg/m² of irinotecan during the I administration of the first therapy cycle. In order to define the pharmacokinetic interactions between CPT-11 and bevacizumab (BV), the pharmacokinetic profile of CPT-11 was evaluated in absence and presence of BV in the same patient. The pharmacokinetic profile of CPT-11 alone was assessed at the first chemotherapy treatment in which BV was administered on day 3 (50 h after the start of CPT-11 infusion). Whereas, irinotecan pharmacokinetics in combination with BV was performed during the second treatment of the first cycle, when BV was administered before CPT-11 dosage.</p

Calibration curve of CPT-11 and its main metabolites SN-38, SN-38G and APC in human plasma.

<p>Calibration curve of CPT-11 and its main metabolites SN-38, SN-38G and APC in human plasma.</p

Metabolic pathway and structures of CPT-11 and its main metabolites: SN-38, SN-38G, APC and NPC.

<p>Cyt P450: cytochrome P450; UGT1A1: UDP-glucoronosyltransferase 1A1.</p

MS/MS mass spectra of CPT-11, SN-38, SN-38G and APC with chemical structures and identification of the main fragment ions.

<p>The fragment ion at <i>m/z</i> *248 of SN-38G is not shown in the MS/MS mass spectrum because it requires, for its formation, a higher collision energy than the other fragments.</p

Ser294 is involved in Ser118 and Ser167 phosphorylation and participates in ERalpha dimerization.

<p>A) 293FT cells were transfected with ERalpha wild-type (W) and Ser294Ala plasmid constructs. After 17-β-estradiol treatment, cell lysates were immunoblotted and probed with ERalpha Ser118 and Ser167 phospho-specific antibodies. The GFP plasmid was utilized as the internal control of transfection. Quantification represents the ratio between the phosphorylated and total ERalpha protein and normalized to WT ERalpha protein. B) The pGL2 Luciferase reporter construct was transfected into 293FT cells together with either GAL4/VP16 ERalpha WT or GAL4/VP16 ERalpha Ser294Ala plasmids. The values represent the luciferase activity normalized to Renilla. Transfected cells were treated with 10 nM 17-β-estradiol to stimulate ERalpha dimerization. The data represent the average of three different experiments. Error bars represent ±SD. p-value<** 0.01 C) Real-time PCR analysis of CTSD and TFF1 ERalpha-target genes in scrambled and Pin1 kd cells. The values are normalized to the GAPDH gene. Data represent the average of three replicates. Error bars represent ±SD. p-value<* 0.05 or ** 0.01.</p

The PI3K-CDK pathway is responsible for interaction between Pin1 and ERalpha.

<p>A) MCF7 cells were grown in normal serum and treated with different micro Molar concentration of LY294002 or flavopiridol drugs for 16 hours. Total protein lysates were pulled down (PD) with GST-Pin1. AKT was used as the control of LY294002 treatment. B) Pin1 and ERalpha working model under basal and estradiol-stimulated conditions. See text for details.</p

Pin1 associates with ERalpha both <i>in vitro</i> and <i>in vivo</i>.

<p>A) Serum-starved cells were treated with estradiol (E2) or ethanol for 45′. Cells were harvested and pellets immunoprecipitated with anti-Pin1, and analyzed by Western blot with anti-ERalpha antibody. Pin1 kd cells or IgG were used as a negative control. B) MCF7 cells were transfected with shRNA scramble (SCR) or shRNA Pin1 (kd1 and kd2), and Pin1 protein level was detected by immunoblotting. GAPDH was used as the load control. Quantification represents the ratio between the Pin1 and GAPDH proteins and normalized to scrambled cells. C) GST, GST-Pin1, GST-WW or GST-PPIase fusion proteins were incubated with ERalpha-positive MCF7 cell lysates and the bound proteins were analyzed by immunoblotting with an ERalpha antibody.</p

ERalpha CD domain interacts with Pin1.

<p>A) Schematic representation of ERalpha domains, showing the predicted prolyl isomerase sites (Ser/Thr-Pro). B) ERalpha cDNA was split into three different domains (AB, CD, EF) and tagged with histidine. GST or GST-Pin1 pull-down assay was performed in 293FT cells and analyzed by Western blot with anti-6×His antibody. C) Site-directed mutagenesis was performed to replace Serine 294 with Alanine on the ERalphaCD domain. Plasmids with wild-type (w) or mutant (m) CD His-tagged domain were transfected in 293FT cells and pulled down with GST-Pin1 protein. Anti-His antibody was used in Western blots to detect the interaction.</p